The overall objective is to understand the structure and function of [Fe- S] proteins. [Fe-S] proteins are ubiquitous in biological systems. They are involved in fundamental biological processes such as enzyme catalysis, electron transfer and regulation of genetic expression. They are metalloproteins containing one or more of a variety of clusters of inorganic elements including iron, sulfur and other hetero-atoms. Fundamental properties of [Fe-S] proteins are not understood, including the function of [Fe-S] clusters in chemical transformations, the biosynthesis of [Fe-S] clusters, the role of [Fe-S] clusters in regulatory proteins, and the control and selection of [Fe-S] cluster reduction potential and oxidation state exerted by protein environments. These issues will be addressed by in-depth study of two [Fe-S] proteins using mutagenesis and crystallography coupled with biochemical, kinetic and spectroscopic experiments. The proteins studied are aconitase and 7Fe ferredoxin. Aconitase employs an [4Fe-4S] cluster to catalyze a (de)hydration reaction and to regulate expression of mRNA. 7Fe ferredoxin employs both [3Fe-4S] and [4Fe-4S] clusters in electron transfer reactions. The opportunity exists for the first time to gain fundamental understanding of the catalytic activity of an [Fe-S] enzyme. In mitochondrial aconitase the specific interactions of active site side chains and the [4Fe-4S] cluster with one substrate and five inhibitors are known, the binding modes of three natural substrates in a three-step reaction are established, and a chemical mechanism is postulated, implying functions for key residues. Experiments to study the structure and function of site-directed mutant enzymes will test, confirm and correct the model for the overall reaction, and probe the function and directionality of specific hydrogen bonds and proton transfers within a network of interactions surrounding three key oxygen atoms in the active site. Site-directed mutants will be used to study essential conformational changes, substrate recognition and binding, and the structures of intermediates in the assembly of the [4Fe-45] cluster. These experiments are directly relevant to cytosolic aconitase, which utilizes [Fe-S] cluster (dis)assembly to regulate mRNA binding. In 7Fe ferredoxin a focused, interdisciplinary study of the structure and function of site- directed mutant proteins will deduce the specific pathway of proton transfer to the reduced [3Fe-45]0 cluster, discover the actors controlling reduction potential of the [4Fe-4S]2+/1+ cluster, and explore the effect of cysteine ligand spatial distribution upon [Fe-S] cluster type and activity. New crystal structures of two [Fe-S] proteins, a dehydratase and a related ferredoxin, and of a functionally related flavoprotein, will provide critical perspective on these specific experiments.